Polypeptides having lipase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having lipase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to isolated polypeptides havinglipase activity and isolated nucleic acid sequences encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

[0003] 2. Description of the Related Art

[0004] Lipases (EC 3.1.1.3) are enzymes that can hydrolyze triglyceridesto release fatty acid.

[0005] Detergents formulated with lipolytic enzymes are known to haveimproved properties for removing fatty stains. For example, LIPOLASE™(Novo Nordisk A/S, Bagsvaerd, Denmark), a microbial lipase obtained fromthe fungus Thermomyces lanuginosus (also called Humicola lanuginosa),has been introduced into many commercial brands of detergent.

[0006] WO 98/26057 discloses a polypeptide having lipase andphospholipase activity (GenBank Acc. No. A85215) obtained from Fusariumoxysporum.

[0007] It is an object of the present invention to provide improvedpolypeptides having lipase activity and nucleic acid encoding thepolypeptides.

SUMMARY OF THE INVENTION

[0008] The present invention relates to isolated polypeptides havinglipase activity selected from the group consisting of:

[0009] (a) a polypeptide having an amino acid sequence which has atleast 85% identity with amino acids 31 to 350 of SEQ ID NO:2;

[0010] (b) a polypeptide encoded by a nucleic acid sequence whichhybridizes under high stringency conditions with (i) nucleotides 1525 to2530 of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides1525 to 2530 of SEQ ID NO:1, (iii) a subsequence of (i) or (ii) of atleast 100 nucleotides, or (iv) a complementary strand of (i), (ii), or(iii);

[0011] (c) a variant of the polypeptide having an amino acid sequence ofSEQ ID NO:2 comprising a substitution, deletion, and/or insertion of oneor more amino acids; and

[0012] (d) a fragment of (a) or (b) that has lipase activity.

[0013] The present invention also relates to isolated nucleic acidsequences encoding the polypeptides and to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIGS. 1A and 1B show the genomic DNA sequence and the deducedamino acid sequence of a Fusarium venenatum lipase (SEQ ID NOS:1 and 2,respectively).

[0015]FIG. 2 shows a restriction map of pSheB1.

[0016]FIG. 3 show a restriction map of pEJG60.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Polypeptides Having Lipase Activity

[0018] The term “lipase activity” is defined herein as a triacylglycerolacylhydrolase activity which catalyzes the hydrolysis of atriacylglycerol to diacylglycerol and a fatty acid anion.

[0019] A substrate for lipase is prepared by emulsifying tributyrin(glycerin tributyrate) using gum Arabic as emulsifier. The hydrolysis oftributyrin at 30° C. at pH 7 is followed in a pH-stat titrationexperiment. One unit of lipase activity (1 LU) equals the amount ofenzyme capable of releasing 1 μmol butyric acid/minute at the standardconditions. 1 KLU=1000 LU. For purposes of the present invention,however, lipase activity is determined by measuring the hydrolysis of 2mM p-nitrophenyl butyrate in 100 mM MOPS pH 7.5, 4 mM CaCl₂, 990 μl ofDMSO, 80 μl of 1% AOS at pH 7.5, 25° C. One unit of lipase activity isdefined as 1.0 μmole of p-nitro phenolate anion produced per minute at25° C., pH 7.5.

[0020] In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 31 to 350 of SEQ ID NO:2 (i.e., the maturepolypeptide) of at least about 85%, preferably at least about 90%, morepreferably at least about 95%, and most preferably at least about 97%,which have lipase activity (hereinafter “homologous polypeptides”). In apreferred embodiment, the homologous polypeptides have an amino acidsequence which differs by five amino acids, preferably by four aminoacids, more preferably by three amino acids, even more preferably by twoamino acids, and most preferably by one amino acid from amino acids 31to 350 of SEQ ID NO:2. For purposes of the present invention, the degreeof identity between two amino acid sequences is determined by theClustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=1,gap penalty=3, windows=5, and diagonals=5.

[0021] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; ora fragment thereof that has lipase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 31 to 350 ofSEQ ID NO:2, or an allelic variant thereof; or a fragment thereof thathas lipase activity. In another preferred embodiment, the polypeptide ofthe present invention comprises amino acids 31 to 350 of SEQ ID NO:2. Inanother preferred embodiment, the polypeptide of the present inventionconsists of the amino acid sequence of SEQ ID NO:2 or an allelic variantthereof; or a fragment thereof that has lipase activity. In anotherpreferred embodiment, the polypeptide of the present invention consistsof the amino acid sequence of SEQ ID NO:2. In another preferredembodiment, the polypeptide consists of amino acids 31 to 350 of SEQ IDNO:2 or an allelic variant thereof; or a fragment thereof that haslipase activity. In another preferred embodiment, the polypeptideconsists of amino acids 31 to 350 of SEQ ID NO:2.

[0022] A fragment of SEQ ID NO:2 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. Preferably, a fragment contains at least 260 aminoacid residues, more preferably at least 280 amino acid residues, andmost preferably at least 300 amino acid residues.

[0023] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0024] In a second embodiment, the present invention relates to isolatedpolypeptides having lipase activity which are encoded by nucleic acidsequences which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with a nucleic acid probe which hybridizes underthe same conditions with (i) nucleotides 1525 to 2530 of SEQ ID NO:1,(ii) the cDNA sequence contained in nucleotides 1525 to 2530 of SEQ IDNO:1, (iii) a subsequence of (i) or (ii), or (iv) a complementary strandof (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.). The subsequence of SEQ ID NO:1 may be at least 100nucleotides or preferably at least 200 nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has lipase activity.The polypeptides may also be allelic variants or fragments of thepolypeptides that have lipase activity.

[0025] The nucleic acid sequence of SEQ ID NO:1 or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO:2 or a fragmentthereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having lipase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

[0026] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA which hybridizes with the probesdescribed above and, which encodes a polypeptide having lipase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1 or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO:1, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

[0027] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO:2, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO:1. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO:1. In anotherpreferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pEJG60 which is contained in Escherichiacoli NRRL B-30333, wherein the nucleic acid sequence encodes apolypeptide having lipase activity. In another preferred embodiment, thenucleic acid probe is the mature polypeptide coding region contained inplasmid pEJG60 which is contained in Escherichia coli NRRL B-30333.

[0028] For long probes of at least 100 nucleotides in length, very lowto very high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0029] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0030] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at about5° C. to about 10° C. below the calculated Tm using the calculationaccording to Bolton and McCarthy (1962, Proceedings of the NationalAcademy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6,6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodiumpyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mgof yeast RNA per ml following standard Southern blotting procedures.

[0031] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6× SCCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSCat 5° C. to 10° C. below the calculated T_(m).

[0032] In a third embodiment, the present invention relates to variantsof the polypeptide having an amino acid sequence of SEQ ID NO:2comprising a substitution, deletion, and/or insertion of one or moreamino acids.

[0033] The amino acid sequences of the variant polypeptides may differfrom the amino acid sequence of SEQ ID NO:2 or the mature polypeptidethereof by an insertion or deletion of one or more amino acid residuesand/or the substitution of one or more amino acid residues by differentamino acid residues. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

[0034] Examples of conservative substitutions are within the group ofbasic amino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

[0035] In a fourth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ IDNO:2 or the mature polypeptide thereof. The immunochemical propertiesare determined by immunological cross-reaction identity tests by thewell-known Ouchterlony double immunodiffusion procedure. Specifically,an antiserum containing polyclonal antibodies which are immunoreactiveor bind to epitopes of the polypeptide having the amino acid sequence ofSEQ ID NO:2 or the mature polypeptide thereof are prepared by immunizingrabbits (or other rodents) according to the, procedure described byHarboe and Ingild, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, AManual of Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 23, or Johnstone and Thorpe, Immunochemistryin Practice, Blackwell Scientific Publications, 1982 (more specificallypages 27-31). A polypeptide having immunochemical identity is apolypeptide which reacts with the antiserum in an identical fashion suchas total fusion of precipitates, identical precipitate morphology,and/or identical electrophoretic mobility using a specificimmunochemical technique. A further explanation of immunochemicalidentity is described by Axelsen, Bock, and Krøll, In N. H. Axelsen, J.Krøll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973,; Chapter10. A polypeptide having partial immunochemical identity is apolypeptide which reacts with the antiserum in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

[0036] The antibody may also be a monoclonal antibody. Monoclonalantibodies may be prepared and used, e.g., according to the methods ofE. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0037] The polypeptides of the present invention have at least 20%,preferably at least 40%, more preferably at least 60%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 100% of the lipase activity of the maturepolypeptide of SEQ ID NO:2.

[0038] A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

[0039] A polypeptide of the present invention may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

[0040] A polypeptide of the present invention may be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

[0041] In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

[0042] In another preferred embodiment, the polypeptide is anAspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

[0043] In another preferred embodiment, the polypeptide is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum polypeptide.

[0044] In a more preferred embodiment, the Fusarium venenatum cell isFusarium venenatum A3/5, which was originally deposited as Fusariumgraminearum ATCC 20334 and recently reclassified as Fusarium venenatumby Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; aswell as taxonomic equivalents of Fusarium venenatum regardless of thespecies name by which they are currently known. In another preferredembodiment, the Fusarium venenatum cell is a morphological mutant ofFusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosedin WO 97/26330.

[0045] It will be understood that for the aforementioned species, theinvention encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents. For example, taxonomicequivalents of Fusarium are defined by D. L. Hawksworth, P. M. Kirk, B.C. Sutton, and D. N. Pegler (editors), 1995, In Ainsworth & Bisby'sDictionary of the Fungi, Eighth Edition, CAB International, UniversityPress, Cambridge, England, pp.173-174.

[0046] Strains of these species are readily accessible to the public ina number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

[0047] Furthermore, such polypeptides may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

[0048] As defined herein, an “isolated” polypeptide is a polypeptidewhich is essentially free of other non-lipase polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

[0049] Polypeptides encoded by nucleic acid sequences of the presentinvention also include fused polypeptides or cleavable fusionpolypeptides in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleic acid sequence (or a portionthereof) encoding another polypeptide to a nucleic acid sequence (or aportion thereof) of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

[0050] Nucleic Acid Sequences

[0051] The present invention also relates to isolated nucleic acidsequences which encode a polypeptide of the present invention. In apreferred embodiment, the nucleic acid sequence is set forth in SEQ IDNO:1. In another more preferred embodiment, the nucleic acid sequence isthe sequence contained in plasmid pEJG60 that is contained inEscherichia coli NRRL B-30333. In another preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region of SEQ IDNO:1. In another more preferred embodiment, the nucleic acid sequence isthe mature polypeptide coding region contained in plasmid pEJG60 that iscontained in Escherichia coli NRRL B-30333. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof,which differ from SEQ ID NO:1 by virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO:1which encode fragments of SEQ ID NO:2 that have lipase activity.

[0052] A subsequence of SEQ ID NO:1 is a nucleic acid sequenceencompassed by SEQ ID NO:1 except that one or more nucleotides from the5′ and/or 3′ end have been deleted. Preferably, a subsequence containsat least 780 nucleotides, more preferably at least 840 nucleotides, andmost preferably at least 900 nucleotides. The present invention alsorelates to mutant nucleic acid sequences comprising at least onemutation in the mature polypeptide coding sequence of SEQ ID NO:1, inwhich the mutant nucleic acid sequence encodes a polypeptide whichconsists of amino acids 31 to 350 of SEQ ID NO:2.

[0053] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used. Thenucleic acid sequence may be cloned from a strain of Fusarium, oranother or related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

[0054] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, CDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

[0055] The present invention also relates to nucleic acid sequenceswhich have a degree of homology to the mature polypeptide codingsequence of SEQ ID NO:1 (i.e., nucleotides 1525 to 2530) of at leastabout 85%, preferably about 90%, more preferably about 95%, and mostpreferably about 97% homology, which encode an active polypeptide. Forpurposes of the present invention, the degree of homology between twonucleic acid sequences is determined by the Wilbur-Lipman method (Wilburand Lipman, 1983, Proceedings of the National Academy of Science USA 80:726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters were Ktuple=3, gap penalty=3, andwindows=20.

[0056] Modification of a nucleic acid sequence encoding a polypeptide ofthe present invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

[0057] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for lipase activity to identifyamino acid residues that are critical to the activity of the molecule.Sites of substrate-enzyme interaction can also be determined by analysisof the three-dimensional structure as determined by such techniques asnuclear magnetic resonance analysis, crystallography or photoaffinitylabelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smithet al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver etal., 1992, FEBS Letters 309: 59-64).

[0058] The present invention also relates to isolated nucleic acidsequences encoding a polypeptide of the present invention, whichhybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with the nucleic acid sequence of SEQ ID NO:1 or itscomplementary strand; or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

[0059] The present invention also relates to isolated nucleic acidsequences produced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) the cDNA sequencecontained in nucleotides 1525 to 2530 of SEQ ID NO:1, (iii) asubsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii),or (iii); and (b) isolating the nucleic acid sequence. The subsequenceis preferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has lipase activity.

[0060] Methods for Producing Mutant Nucleic Acid Sequences

[0061] The present invention further relates to methods for producing amutant nucleic acid sequence, comprising introducing at least onemutation into the mature polypeptide coding sequence of SEQ ID NO:1 or asubsequence thereof, wherein the mutant nucleic acid sequence encodes apolypeptide which consists of amino acids 31 to 350 of SEQ ID NO:2 or afragment thereof which has lipase activity.

[0062] The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

[0063] Nucleic Acid Constructs

[0064] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Expression will be understood to include anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

[0065] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid combined and juxtaposed in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a nucleic acid sequence which directly specifies the aminoacid sequence of its protein product. The boundaries of a genomic codingsequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

[0066] An isolated nucleic acid sequence encoding a polypeptide of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

[0067] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

[0068] The control sequence may be an appropriate promoter sequence, anucleic acid sequence which is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscriptional control sequences which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

[0069] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xyIA and xyIBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0070] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention in a filamentousfungal host cell are promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787),as well as the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

[0071] In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

[0072] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

[0073] Preferred terminators for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

[0074] Preferred terminators for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

[0075] The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

[0076] Preferred leaders for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

[0077] Suitable leaders for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0078] The control sequence may also be a polyadenylation sequence, asequence operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

[0079] Preferred polyadenylation sequences for filamentous fungal hostcells are obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

[0080] Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

[0081] The control sequence may also be a signal peptide coding regionthat codes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

[0082] Effective signal peptide coding regions for bacterial host cellsare the signal peptide coding regions obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0083] Effective signal peptide coding regions for filamentous fungalhost cells are the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0084] In a preferred embodiment, the signal peptide coding region isnucleotides 1376 to 1420 of SEQ ID NO:1 which encode amino acids 1 to 15of SEQ ID NO:2.

[0085] Useful signal peptides for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions aredescribed by Romanos et al., 1992, supra.

[0086] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

[0087] In a preferred embodiment, the propeptide coding region isnucleotides 1421 to 1465 of SEQ ID NO:1 which encode amino acids 16 to30 of SEQ ID NO:2.

[0088] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0089] It may also be desirable to add regulatory sequences which allowthe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

[0090] The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

[0091] In both embodiments, the introduction of these components resultsin production of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

[0092] The targeting sequence can be within the endogenous gene,immediately adjacent to the gene, within an upstream gene, or upstreamof and at a distance from the endogenous gene. One or more targetingsequences can be used. For example, a circular plasmid or DNA fragmentpreferably employs a single targeting sequence, while a linear plasmidor DNA fragment preferably employs two targeting sequences.

[0093] The regulatory sequence of the construct can be comprised of oneor more promoters, enhancers, scaffold-attachment regions or matrixattachment sites, negative regulatory elements, transcription bindingsites, or combinations of these sequences.

[0094] The constructs further contain one or more exons of theendogenous gene. An exon is defined as a DNA sequence which is copiedinto RNA and is present in a mature mRNA molecule such that the exonsequence is in-frame with the coding region of the endogenous gene. Theexons can, optionally, contain DNA which encodes one or more amino acidsand/or partially encodes an amino acid. Alternatively, the exon containsDNA which corresponds to a 5′ non-encoding region. Where the exogenousexon or exons encode one or more amino acids and/or a portion of anamino acid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

[0095] The splice-donor site of the constructs directs the splicing ofone exon to another exon. Typically, the first exon lies 5′ of thesecond exon, and the splice-donor site overlapping and flanking thefirst exon on its 3′ side recognizes a splice-acceptor site flanking thesecond exon on the 5′ side of the second exon. A splice-acceptor site,like a splice-donor site, is a sequence which directs the splicing ofone exon to another exon. Acting in conjunction with a splice-donorsite, the splicing apparatus uses a splice-acceptor site to effect theremoval of an intron.

[0096] Expression Vectors

[0097] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals. Thevarious nucleic acid and control sequences described above may be joinedtogether to produce a recombinant expression vector which may includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the polypeptide atsuch sites. Alternatively, the nucleic acid, sequence of the presentinvention may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

[0098] The recombinant expression vector may be any vector (e.g., aplasmid or virus) which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

[0099] The vector may be an autonomously replicating vector, i.e., avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

[0100] The vectors of the present invention preferably contain one ormore selectable markers which permit easy selection of transformedcells. A selectable marker is a gene the product of, which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

[0101] The vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

[0102] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

[0103] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe host cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,and pAMβ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes its functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1433).

[0104] More than one copy of a nucleic acid sequence of the presentinvention may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

[0105] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors of the present inventionare well known to one skilled in the art (see, e.g., Sambrook et al.,1989, supra).

[0106] Host Cells

[0107] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

[0108] The host cell may be a unicellular microorganism, e.g., aprokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

[0109] Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

[0110] The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0111] The host cell may be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

[0112] In a preferred embodiment, the host cell is a fungal cell.“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

[0113] In a more preferred embodiment, the fungal host cell is a yeastcell. “Yeast” as used herein includes ascosporogenous yeast(Endomycetales), basidiosporogenous yeast, and yeast belonging to theFungi Imperfecti (Blastomycetes). Since the classification of yeast maychange in the future, for the purposes of this invention, yeast shall bedefined as described in Biology and Activities of Yeast (Skinner, F. A.,Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol.Symposium Series No. 9, 1980).

[0114] In an even more preferred embodiment, the yeast host cell is aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

[0115] In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

[0116] In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are generally characterizedby a mycelial wall composed of chitin, cellulose, glucan, chitosan,mannan, and other complex polysaccharides. Vegetative growth is byhyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

[0117] In an even more preferred embodiment, the filamentous fungal hostcell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, or Trichoderma.

[0118] In a most preferred embodiment, the filamentous fungal host cellis an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0119] Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

[0120] Methods of Production

[0121] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce the polypeptide; and (b) recovering thepolypeptide. Preferably, the strain is of the genus Fusarium, and morepreferably Fusarium venenatum.

[0122] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

[0123] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the mature polypeptide coding region of SEQ IDNO:1, wherein the mutant nucleic acid sequence encodes a polypeptidewhich consists of amino acids 31 to 350 of SEQ ID NO:2, and (b)recovering the polypeptide.

[0124] The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulatory sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670.

[0125] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0126] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0127] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

[0128] The polypeptides of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

[0129] Plants

[0130] The present invention also relates to a transgenic plant, plantpart, or plant cell which has been transformed with a nucleic acidsequence encoding a polypeptide having lipase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and Theologicalproperties, or to destroy an antinutritive factor.

[0131] The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). Examples of monocot plants are grasses,such as meadow grass (blue grass, Poa), forage grass such as festuca,lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat,oats, rye, barley, rice, sorghum, and maize (corn).

[0132] Examples of dicot plants are tobacco, legumes, such as lupins,potato, sugar beet, pea, bean and soybean, and cruciferous plants(family Brassicaceae), such as cauliflower, rape seed, and the closelyrelated model organism Arabidopsis thaliana.

[0133] Examples of plant parts are stem, callus, leaves, root, fruits,seeds, and tubers. Also specific plant tissues, such as chloroplast,apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm areconsidered to be a plant part. Furthermore, any plant cell, whatever thetissue origin, is considered to be a plant part.

[0134] Also included within the scope of the present invention are theprogeny of such plants, plant parts and plant cells.

[0135] The transgenic plant or plant cell expressing a polypeptide ofthe present invention may be constructed in accordance with methodsknown in the art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

[0136] Conveniently, the expression construct is a nucleic acidconstruct which comprises a nucleic acid sequence encoding a polypeptideof the present invention operably linked with appropriate regulatorysequences required for expression of the nucleic acid sequence in theplant or plant part of choice. Furthermore, the expression construct maycomprise a selectable marker useful for identifying host cells intowhich the expression construct has been integrated and DNA sequencesnecessary for introduction of the construct into the plant in question(the latter depends on the DNA introduction method to be used).

[0137] The choice of regulatory sequences, such as promoter andterminator sequences and optionally signal or transit sequences isdetermined, for example, on the basis of when, where, and how thepolypeptide is desired to be expressed. For instance, the expression ofthe gene encoding a polypeptide of the present invention may beconstitutive or inducible, or may be developmental, stage or tissuespecific, and the gene product may be targeted to a specific tissue orplant part such as seeds or leaves. Regulatory sequences are, forexample, described by Tague et al., 1988, Plant Physiology 86: 506.

[0138] For constitutive expression, the 35S-CaMV promoter may be used(Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters maybe, for example, a promoter from storage sink tissues such as seeds,potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu etal.,1993, Plant Molecular Biology 22: 573-588).

[0139] A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

[0140] The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

[0141] The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0142] Presently, Agrobacterium tumefaciens-mediated gene transfer isthe method of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

[0143] Following transformation, the transformants having incorporatedtherein the expression construct are selected and regenerated into wholeplants according to methods well-known in the art.

[0144] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having lipase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[0145] Removal or Reduction of Lipase Activity

[0146] The present invention also relates to methods for producing amutant cell of a parent cell, which comprises disrupting or deleting anucleic acid sequence encoding the polypeptide or a control sequencethereof, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

[0147] The construction of strains which have reduced lipase activitymay be conveniently accomplished by modification or inactivation of anucleic acid sequence necessary for expression of the polypeptide havinglipase activity in the cell. The nucleic acid sequence to be modified orinactivated may be, for example, a nucleic acid sequence encoding thepolypeptide or a part thereof essential for exhibiting lipase activity,or the nucleic acid sequence may have a regulatory function required forthe expression of the polypeptide from the coding sequence of thenucleic acid sequence. An example of such a regulatory or controlsequence may be a promoter sequence or a functional part thereof, i.e.,a part which is sufficient for affecting expression of the polypeptide.Other control sequences for possible modification are described above.

[0148] Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting orscreening for cells in which the lipase producing capability has beenreduced. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

[0149] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues.

[0150] When such agents are used, the mutagenesis is typically performedby incubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced lipase activity or production.

[0151] Modification or inactivation of production of a polypeptide ofthe present invention may be accomplished by introduction, substitution,or removal of one or more nucleotides in the nucleic acid sequenceencoding the polypeptide or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change of the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleicacid sequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

[0152] An example of a convenient way to eliminate or reduce productionby a host cell of choice is by gene replacement or gene interruption. Inthe gene interruption method, a nucleic acid sequence corresponding tothe endogenous gene or gene fragment of interest is mutagenized in vitroto produce a defective nucleic acid sequence which is then transformedinto the host cell to produce a defective gene. By homologousrecombination, the defective nucleic acid sequence replaces theendogenous gene or gene fragment. It may be desirable that the defectivegene or gene fragment also encodes a marker which may be used forselection of transformants in which the gene encoding the polypeptidehas been modified or destroyed.

[0153] Alternatively, modification or inactivation of the nucleic acidsequence may be performed by established anti-sense techniques using anucleotide sequence complementary to the polypeptide encoding sequence.More specifically, production of the polypeptide by a cell may bereduced or eliminated by introducing a nucleotide sequence complementaryto the nucleic acid sequence encoding the polypeptide which may betranscribed in the cell and is capable of hybridizing to the polypeptidemRNA produced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the polypeptide mRNA, theamount of polypeptide translated is thus reduced or eliminated.

[0154] It is preferred that the cell to be modified in accordance withthe methods of the present invention is of microbial origin, forexample, a fungal strain which is suitable for the production of desiredprotein products, either homologous or heterologous to the cell.

[0155] The present invention further relates to a mutant cell of aparent cell which comprises a disruption or deletion of a nucleic acidsequence encoding the polypeptide or a control sequence thereof, whichresults in the mutant cell producing less of the polypeptide than theparent cell.

[0156] The polypeptide-deficient mutant cells so created areparticularly useful as host cells for the expression of homologousand/or heterologous polypeptides. Therefore, the present inventionfurther relates to methods for producing a homologous or heterologouspolypeptide comprising (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

[0157] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of lipase activity byfermentation of a cell which produces both a polypeptide of the presentinvention as well as the protein product of interest by adding aneffective amount of an agent capable of inhibiting lipase activity tothe fermentation broth before, during, or after the fermentation hasbeen completed, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

[0158] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of lipase activity bycultivating the cell under conditions permitting the expression of theproduct, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the lipase activity substantially,and recovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with a lipase inhibitor.

[0159] In accordance with this aspect of the invention, it is possibleto remove at least 60%, preferably at least 75%, more preferably atleast 85%, still more preferably at least 95%, and most preferably atleast 99% of the lipase activity. Complete removal of lipase activitymay be obtained by use of this method.

[0160] The combined pH and temperature treatment is preferably carriedout at a pH in the range of 6.5-7 and a temperature in the range of25-40° C. for a sufficient period of time to attain the desired effect,where typically, 30 to 60 minutes is sufficient.

[0161] The methods used for cultivation and purification of the productof interest may be performed by methods known in the art.

[0162] The methods of the present invention for producing an essentiallylipase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thelipase-deficient cells may also be used to express heterologous proteinsof pharmaceutical interest such as hormones, growth factors, receptors,and the like.

[0163] It will be understood that the term “eukaryotic polypeptides”includes not only native polypeptides, but also those polypeptides,e.g., enzymes, which have been modified by amino acid substitutions,deletions or additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

[0164] In a further aspect, the present invention relates to a proteinproduct essentially free from lipase activity which is produced by amethod of the present invention.

[0165] Compositions

[0166] In a still further aspect, the present invention relates tocompositions comprising a polypeptide of the present invention.Preferably, the compositions are enriched in a polypeptide of thepresent invention. In the present context, the term “enriched” indicatesthat the lipase activity of the composition has been increased, e.g.,with an enrichment factor of 1.1.

[0167] The composition may comprise a polypeptide of the invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be producible by means of amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillusoryzae, or Trichoderma, Humicola, preferably Humicola insolens, orFusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum.

[0168] The polypeptide compositions may be prepared in accordance withmethods known in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

[0169] Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

[0170] Uses

[0171] The present invention is also directed to methods for using thepolypeptides having lipase activity in any industrial application oflipases, e.g., in detergents.

[0172] Use in Detergent

[0173] The variant may be used as a detergent additive, e.g., at aconcentration (expressed as pure enzyme protein) of 0.001-10 (e.g.,0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg perliter of wash liquor.

[0174] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations. In a laundry detergent, the variant may beeffective for the removal of fatty stains, for whiteness maintenance andfor dingy cleanup. A laundry detergent composition may be formulated asdescribed in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939and WO 97/43375.

[0175] The detergent composition of the invention may particularly beformulated for hand or machine dishwashing operations e.g., as describedin GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble). In adishwashing composition, the variant may be effective for removal ofgreasy/oily stains, for prevention of the staining/discoloration of thedishware and plastic components of the dishwasher by highly coloredcomponents and the avoidance of lime soap deposits on the dishware.

[0176] Use in Degumming

[0177] A polypeptide of the present invention may be used for degummingan aqueous carbohydrate solution or slurry to improve its filterability,particularly, a starch hydrolysate, especially a wheat starchhydrolysate which is difficult to filter and yields cloudy filtrates.The treatment may be performed using methods well known in the art. See,for example, EP 219,269 and EP 808,903.

[0178] Signal Peptide and Propeptide

[0179] The present invention also relates to nucleic acid constructscomprising a gene encoding a protein operably linked to one or both of afirst nucleic acid sequence consisting of nucleotides 1376 to 1429 ofSEQ ID NO:1 encoding a signal peptide consisting of amino acids 1 to 15of SEQ ID NO:2 and a second nucleic acid sequence consisting ofnucleotides 1421 to 1465 of SEQ ID NO:1 encoding a propeptide consistingof amino acids 16 to 30 of SEQ ID NO:2, wherein the gene is foreign tothe first and second nucleic acid sequences.

[0180] The present invention also relates to recombinant expressionvectors and recombinant host cells comprising such nucleic acidconstructs.

[0181] The present invention also relates to methods for producing aprotein comprising (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

[0182] The first and second nucleic acid sequences may be operablylinked to foreign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As noted earlier, where both signal peptide-andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

[0183] The protein may be native or heterologous to a host cell. Theterm “protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

[0184] Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred embodiment, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred embodiment, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

[0185] The gene may be obtained from any prokaryotic, eukaryotic, orother source.

[0186] The present invention is further described by the followingexamples which should not be construed as limiting the scope of theinvention.

EXAMPLES

[0187] Chemicals used as buffers and substrates were commercial productsof at least reagent grade.

[0188] Strains

[0189]Fusarium venenatum WTY700 3.8d, a spore-purified tri5-minus,dps1-minus strain, was used as the recipient strain for transformationexperiments. Fusarium venenatum WTY700 3.8d is a morphological mutant ofFusarium venenatum strain ATCC 20334 (Wiebe et al., 1991, Mycol.Research 95: 1284-1288),

Example 1 Fermentation and Mycelial Tissue

[0190]Fusarium venenatum WTY700 3.8d was grown in a two-liter lab-scalefermentor using a fed-batch fermentation scheme with NUTRIOSE™ (RoquetteFreres, S. A., Beinheim, France) as the carbon source and yeast extract.Ammonium phosphate was provided in the feed. The fermentation wasmaintained at pH 6-6.5 and 30 ° C. with positive dissolved oxygen.

[0191] Mycelial samples were harvested at 2, 4, 6, and 8 dayspost-inoculum and quick-frozen in liquid nitrogen. The samples werestored at −80° C. until they were disrupted for RNA extraction.

Example 2 cDNA Library Construction

[0192] Total cellular RNA was extracted from the mycelial samplesdescribed in Example 1 according to the method of Timberlake and Barnard(1981, Cell 26: 29-37), and the RNA samples were analyzed by Northernhybridization after blotting from 1% formaldehyde-agarose gels (Davis etal., 1986, Basic Methods in Molecular Biology, Elsevier SciencePublishing Co., Inc., New York). Polyadenylated mRNA fractions wereisolated from total RNA with an mRNA Separator Kit™ (ClontechLaboratories, Inc., Palo Alto, Calif.) according to the manufacturer'sinstructions. Double-stranded cDNA was synthesized, using approximately5 μg of poly(A)+mRNA according to the method of Gubler and Hoffman(1983, Gene 25: 263-269) except a NotI-(dT)18 primer (Pharmacia Biotech,Inc., Piscataway, N.J.) was used to initiate first strand synthesis. ThecDNA was treated with mung bean nuclease (Boehringer MannheimCorporation, Indianapolis, Ind.) and the ends were made blunt with T4DNA polymerase (New England Biolabs, Beverly, Mass.).

[0193] The cDNA was digested with NotI, size selected by agarose gelelectrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (InvitrogenCorporation, Carlsbad, Calif.) which had been cleaved with NotI plusEcoRV and dephosphorylated with calf-intestine alkaline phosphatase(Boehringer Mannheim Corporation, Indianapolis, Ind.). The ligationmixture was used to transform competent E. coli TOP10 cells (InvitrogenCorporation, Carlsbad, Calif.). Transformants were selected on 2YT agarplates (Miller, 1992, A Short Course in Bacterial Genetics. A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.) which contained kanamycinat a final concentration of 50 μg/ml.

Example 3 Template Preparation and Nucleotide Sequencing

[0194] From the cDNA library described in Example 2, 1192 transformantcolonies were picked directly from the transformation plates into96-well microtiter dishes which contained 200 μl of 2YT broth (Miller,1992, supra) with 50 μg/ml kanamycin. The plates were incubatedovernight at 37° C. without shaking. After incubation 100 μl of sterile50% glycerol was added to each well. The transformants were replicatedinto secondary, deep-dish 96-well microculture plates (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) containing 1 ml ofMagnificent Broth™ (MacConnell Research, San Diego, Calif.) supplementedwith 50 μg of kanamycin per ml in each well. The primary microtiterplates were stored frozen at −80° C. The secondary deep-dish plates wereincubated at 37° C. overnight with vigorous agitation (300 rpm) onrotary shaker. To prevent spilling and cross-contamination, and to allowsufficient aeration, each secondary culture plate was covered with apolypropylene pad (Advanced Genetic Technologies Corporation,Gaithersburg, Md.) and a plastic microtiter dish cover.

[0195] DNA was isolated from each well using the 96-well Miniprep Kitprotocol of Advanced Genetic Technologies Corporation (Gaithersburg,Md.) as modified by Utterback et al. (1995, Genome Sci. Technol. 1:1-8). Single-pass DNA sequencing (EST) was done with a Perkin-ElmerApplied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and the reverse lac sequencing primer.

Example 4 Analysis of DNA Sequence Data

[0196] Nucleotide sequence data were scrutinized for quality, andsamples giving improper spacing or ambiguity levels exceeding 3% werediscarded or re-run. Vector sequences and ambiguous base calls at theends of the DNA sequences were trimmed with assistance of FACTURA™software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif. Allsequences were compared to each other to determine multiplicity usingAutoAssembler™ software (Perkin-Elmer Applied Biosystems, Inc., FosterCity, Calif.). Lastly, all sequences were translated in three frames andsearched against a non-redundant database (NRDB) using GeneAssist™software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.)with a modified Smith-Waterman algorithm using the BLOSUM 62 matrix witha threshold score of 70. The NRDB was assembled from Genpept,Swiss-Prot, and PIR databases.

Example 5 Identification of Lipase 1 cDNA Clones

[0197] Putative lipase clones were identified by comparing the deducedamino acid sequence of the ESTs to protein sequences deposited inpublicly available databases such as Swissprot, Genpept, and PIR using amodified Smith-Waterman search algorithm (Perkin-Elmer AppliedBiosystems, Foster City, Calif.). Tentative identification was based onamino acid sequence similarity to numerous fungal lipases. One clone,Fusarium venenatum EST FA0726, was selected for nucleotide sequenceanalysis which revealed that the cDNA clone was truncated at its 5 primeend.

Example 6 Fusarium Venenatum Genomic DNA Extraction

[0198]Fusarium venenatum WTY700 was grown for 24 hours at 28° C. and 150rpm in 25 ml of YEG medium composed per liter of 5 g of yeast extractand 20 g of glucose. Mycelia were then collected by filtration throughMiracloth (Calbiochem, La Jolla, Calif.) and washed once with 25 ml of10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer was drained from themycelia which were subsequently frozen in liquid nitrogen. The frozenmycelia were ground to a fine powder in an electric coffee grinder, andthe powder was added to 20 ml of TE buffer and 5 ml of 20% w/v sodiumdodecylsulfate (SDS) in a disposable plastic centrifuge tube. Themixture was gently inverted several times to ensure mixing, andextracted twice with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to givea final concentration of 0.3 M and the nucleic acids were precipitatedwith 2.5 volumes of ice cold ethanol. The tube was centrifuged at15,000× g for 30 minutes and the pellet was allowed to air dry for 30minutes before resuspension in 0.5 ml of TE buffer. DNase-freeribonuclease A was added to a concentration of 100 μg/ml and the mixturewas incubated at 37° C. for 30 minutes. Proteinase K (200 μg/ml) wasthen added and the mixture was incubated an additional hour at 37° C.Finally, the mixture was extracted twice with phenol:chloroform:isoamylalcohol (25:24:1 v/v/v) before precipitating the DNA with sodium acetateand ethanol according to standard procedures. The DNA pellet was driedunder vacuum, resuspended in TE buffer, and stored at 4° C.

Example 7 Genomic DNA Library Construction, Screening, and Isolation ofGenomic Lipase 1 Clone

[0199] Genomic libraries of Fusarium venenatum WTY700 were constructedin λZipLox according to the manufacturer's instructions (LifeTechnologies, Gaithersburg, Md.). Fusarium venenatum genomic DNA waspartially digested with Tsp5091 and size-fractionated on 1% agarosegels. DNA fragments migrating in the size range 3-7 kb were excised andeluted from the agarose gel slices using Prep-a-Gene reagents (BioRad,Hercules, Calif.). The eluted DNA fragments were ligated withEcoRI-cleaved and dephosphorylated λZipLox vector arms (LifeTechnologies, Gaithersburg, Md.), and the ligation mixtures werepackaged using commercial packaging extracts (Stratagene, La Jolla,Calif.). The packaged DNA libraries were plated and amplified in E. coliY1090ZL cells.

[0200] The cDNA from Fusarium venenatum clone FA0726 was excised fromthe vector plasmid by digestion with EcoRI and NotI yielding anapproximately 900 bp fragment. The fragment was purified by gelelectrophoresis, and radiolabeled with α[³²P] dCTP using a Prime-itRandom Primer Labeling Kit (Stratagene, La Jolla, Calif.).

[0201] Approximately 40,000 plaques from the library were screened byplaque-hybridization (Davis et al., 1980, supra) with the radiolabeledprobe fragment of the Fusarium venenatum lipase gene using highstringency conditions at 45° C. (high stringency=50% formamide, 5× SSPE,0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA). Plaques,which gave hybridization signals, were purified once in E. coli DH10Bcells, and the individual clones were subsequently excised from theλZipLox vector as pZL1-derivatives (D'Alessio et al., 1992, Focus® 14:7).

[0202] One plaque was identified that hybridized strongly to theFusarium venenatum lipase gene probe, and was subsequently excised fromthe XZipLox vector as a pZL1-derivative (D'Alessio et al., 1992, supra).Plasmid DNA was isolated from the clone by passage through E. coli DH10Bcells (Life Technologies, Gaithersburg, Md.) according to themanufacturer's instructions. This clone was designated E. coliDH10B-pFvLipase1.

Example 8 Characterization of the Fusarium Venenatum Genomic CloneEncoding Lipase 1

[0203] DNA sequencing was performed on an Perkin-Elmer Biosystems Model377 XL Automated DNA Sequencer using dye-terminator chemistry (Gieseckeet al., 1992, Journal of Virology Methods 38: 47-60). Contig sequenceswere generated using a transposon insertion strategy (Primer IslandTransposition Kit, Perkin-Elmer/Applied Biosystems, Inc., Foster City,Calif.). The 2.94 kb genomic fragment was sequenced to an averageredundancy of 4.8.

[0204] The nucleotide sequence and deduced amino acid sequence are shownin FIG. 1. The insert contains an open reading frame of 1.153 kbencoding a polypeptide of 350 amino acids. Using the SignalP softwareprogram (Nielsen et al., 1997, Protein Engineering 10: 1-6), a signalpeptide of 15 residues was predicted. The predicted signal peptide isfollowed by a 15 residue propeptide ending with a concanical propeptideGlu/Arg cleavage site. N-terminal sequencing of the lipase 1 proteinsupports this propeptide cleavage site prediction. The open readingframe is interrupted by two introns of 49 bp and 58 bp. Thus, the matureFusarium venenatum lipase comprises 319 amino acids and a predictedmolecular weight of 33.6 kDa. There are 2 potential N-linkedglycosylation sites (Asn-X-Ser/Thr) within Fusarium venenatum lipase 1.

[0205] A comparative alignment of lipase sequences using the Clustal Walgorithm in the Megalign program of DNA-Star, showed that the deducedamino acid sequence of the Fusarium venenatum lipase 1 gene shares 81%identity to the deduced amino acid sequence of a Fusarium oxysporumphospholipase A (EP0869167).

Example 9 Construction of Plasmid pSheB1

[0206] The Fusarium venenatum expression vector pSheB1 (FIG. 2) wasgenerated by modification of pDM181 (WO 98/20136). The modificationsincluded (a) removal of two NcoI sites within the pDM181 sequence, and(b) restoration of the natural translation start of the Fusariumoxysporum trypsin promoter (reconstruction of an NcoI site at the ATGstart codon).

[0207] Removal of two NcoI sites within the pDM181 sequence wasaccomplished using the QuikChange™ site-directed mutagenesis kit(Stratagene Cloning Systems, La Jolla, Calif.) according to themanufacturer's instruction with the following pairs of mutagenesisprimers: 5′-dCAGTGAATTGGCCTCGATGGCCGCGGCCGCGAATT-3′ plus (SEQ ID NO:3)5′-dAATTCGCGGCCGCGGCCATCGAGGCCAATTCACTG-3′ (SEQ ID NO:4)5′-dCACGAAGGAAAGACGATGGCTTTCACGGTGTCTG-3′ plus (SEQ ID NO:5)5′-dCAGACACCGTGAAAGCCATCGTCTTTCCTTCGTG-3′ (SEQ ID NO:6)

[0208] Restoration of the natural translation start of the Fusariumoxysporum trypsin promoter was also accomplished using the StratageneQuikChange™ site directed mutagenesis kit in conjunction with thefollowing pair of mutagenesis primers:5′-dCTATCTCTTCACCATGGTACCTTAATTAAATACCTTGTTGGAAGCG-3′ plus (SEQ ID NO:7)5′-dCGCTTCCAACAAGGTATTTAATTAAGGTACCATGGTGAAGAGATAG-3′ (SEQ ID NO:8)

[0209] All site-directed changes were confirmed by DNA sequence analysisof the appropriate vector regions.

Example 10 Construction of Expression Vector pEJG60

[0210] The lipase-expression vector, pEJG60 was constructed as follows.The lipase coding region was amplified from pFvlipase1 using thefollowing pair of primers: Primer 990658:5′-CGTTCTTTGTCTGTCAGCATGCATCTCCTATCACTCC-3′ (SEQ ID NO:9) Primer 990661:5′-CCAGAGTTTTTGTTATGGTTAATTAATATCGTTACTGCGTAAATG-3′ (SEQ ID NO:10)

[0211] The forward primer introduces a SphI site which contains the ATG,and the reverse primer introduces a PacI site after the stop codon.

[0212] The amplification reaction (100 μl) contained the followingcomponents: 0.5 μg of genomic clone, pFvLipase1, 50 pmol of the forwardprimer, 50 pmol of the reverse primer, 10 mM dNTPs (dATP, dCTP, dGTP,and dTTP), 1× Pwo DNA polymerase buffer, and 2.5 units of Pwo DNApolymerase (Boehringer Mannheim, Indianapolis, Ind.). The reactions wereincubated in a Perkin-Elmer Model 480 Thermal Cycler programmed for 1cycles at 95° C. for 2 minutes; 10 cycles at 94° C. for 45 seconds, 55°C. for 45 seconds, and 72° C. for 2 minutes; 17 cycles at 94° C. for 45seconds, 55° C. for 45 seconds, and 72° C. for 2 minutes with anextension of 20 seconds per cycle; 1 cycle at 72° C. for 10 minutes; anda soak cycle at 4° C. The reaction products were isolated on a 1%agarose gel where a 1.15 kb product band was excised from the gel andpurified using Qiaquik Gel Extraction Kit (Qiagen, Chatsworth, Calif.)according to the manufacturer's instructions.

[0213] The generated fragment was digested with SphI, blunted withKlenow, digested with PacI, and purified by agarose gel electrophoresisand Qiaquik Gel Extraction Kit (Qiagen, Chatsworth, Calif.). Thepurified DNA segment was ligated into pSheB1 (FIG. 2) which waspreviously NcoI digested, treated with DNA polymerase I (Klenowfragment), and digested with PacI. The treatment of the NcoI-digestedvector with Klenow fragment resulted in a filling in of the NcoIcohesive end, thereby making it blunt and compatible with the blunt siteof the lipase DNA segment. The resulting expression plasmid wasdesignated pEJG60 (FIG. 3). The PCR-amplified lipase gene segment wasre-sequenced to verify the absence of any errors.

Example 10 Transformation of Fusarium Venenatum and Analysis of FusariumVenenatum Transformants

[0214] Spores of Fusarium venenatum WTY700 were generated by inoculatinga flask containing 500 ml of RA sporulation medium with 10 plugs from a1× Vogels medium plate (2.5% Noble agar) supplemented with 2.5% glucoseand 2.5 mM sodium nitrate and incubating at 28° C., 150 rpm for 2 to 3days. Spores were harvested through Miracloth (Calbiochem, San Diego,Calif.) and centrifuged 20 minutes at 7000 rpm in a Sorvall RC-5Bcentrifuge (E. I. DuPont De Nemours and Co., Wilmington, Del.). Pelletedspores were washed twice with sterile distilled water, resuspended in asmall volume of water, and then counted using a hemocytometer.

[0215] Protoplasts were prepared by inoculating 100 ml of YEPG mediumwith 4×10⁷ spores of Fusarium venenatum WTY700 and incubating for 16hours at 24° C. and 150 rpm. The culture was centrifuged for 7 minutesat 3500 rpm in a Sorvall RT 6000D (E. I. DuPont De Nemours and Co.,Wilmington, Del.). Pellets were washed twice with 30 ml of 1 M MgSO₄ andresuspended in 15 ml of 5 mg/ml of NOVOZYME 234™ (batch PPM 4356, NovoNordisk A/S, Bagsvaerd, Denmark) in 1 M MgSO₄. Cultures were incubatedat 24° C. and 150 rpm until protoplasts formed. A volume of 35 ml of 2 Msorbitol was added to the protoplast digest and the mixture wascentrifuged at 2500 rpm for 10 minutes. The pellet was resuspended,washed twice with STC, and centrifuged at 2000 rpm for 10 minutes topellet the protoplasts. Protoplasts were counted with a hemocytometerand resuspended in an 8:2:0.1 solution of STC:SPTC:DMSO to a finalconcentration of 1.25×10⁷ protoplasts/ml. The protoplasts were stored at−80° C., after controlled-rate freezing in a Nalgene Cryo 1° C. FreezingContainer (VWR Scientific, Inc., San Francisco, Calif.).

[0216] Frozen protoplasts of Fusarium venenatum WTY700 were thawed onice. Five 1 μg of pEJG60 described in Example 10 and 5 μl of heparin (5mg per ml of STC) was added to a 50 ml sterile polypropylene tube. Onehundred μl of protoplasts was added, mixed gently, and incubated on icefor 30 minutes. One ml of SPTC was added and incubated 20 minutes atroom temperature. After the addition of 25 ml of 40° C. COVE topagarose, the mixture was poured onto an empty 150 mm diameter plate andincubated overnight at room temperature. Then an additional 25 ml of 40°C. COVE top agarose containing 10 mg of BASTA™ per ml was poured on topof the plate and incubated at room temperature for up to 14 days. Theactive ingredient in the herbicide BASTA™ is phosphinothricin. BASTA™was obtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and wasextracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), andonce with chloroform:isoamyl alcohol (24:1) before use.

[0217] Twenty-four transformants were picked directly from the selectionplates (COVE underlay with COVE-BASTA™ overlay) and inoculated into 125ml shake flasks containing 25 ml of M400Da medium supplemented with 1 mMCaCl₂ and 100 μg/ml ampicillin (to prevent bacterial contamination) andincubated at 28° C., 200 rpm on a platform shaker for 7 days. Theuntransformed recipient strain was also included as a negative control.

[0218] Flasks were sampled at 5 and 7 days and assayed for lipaseactivity as described below. The samples were also submitted to SDS-PAGEusing Novex gradient gels (Novex Experimental Technology, San Diego,Calif.).

[0219] Lipase activity was determined as follows: 100 μl of substrate(3.92 ml of 100 mM MOPS pH 7.5, 4 mM CaCl₂, 990 μl of DMSO, 80 μl of 1%AOS, and 20 μl of p-nitrophenyl butyrate) was added to 100 μl of dilutedsample. The samples were diluted accordingly in 100 mM MOPS pH 7.5, 4 mMCaCl₂. The absorbance at 405 nm was monitored for 3 minutes at roomtemperature in a 96-well microtiter plate using a Molecular DevicesThermomax Microplate Reader.

[0220] The lipase assay results indicated that at both 5 and 7 days,most of the transformants produced lipase activity well above that ofthe untransformed control. Shake flask culture broths from transformants#1 and #3, the two highest scorers in the lipase assay, were analyzed ona 16% tricine gel. A prominent polypeptide at a apparent molecularweight of 32-33 kD was observed at both time points and for eachtransformant harboring pEJG60.

Deposit of Biological Material

[0221] The following biological material has been deposited under theterms of the Budapest Treaty with the Agricultural Research ServicePatent Culture Collection, Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, and given the followingaccession number: Deposit Accession Number Date of Deposit E. coilpEJG60 NRRL B-30333 August 22, 2000

[0222] The strain has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0223] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0224] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 10 1 2940 DNA Fusarium venenatum 1 aattcatgtg aatctactat gtaacagtatgttgtattgc attacccatc aacgttgaat 60 cgttgcgacg taacggcccg gttcaagcgagatgtagata tgttggtagt taattgatgg 120 gttaggtatt cttttcatca actcggtattctcattcccc agatatcggc acttgtcttt 180 actccagatt tcatatcgca tcgagttatatacagtccca attgagtcga ctaccccgtc 240 caaaacaggt tttctcacaa accaaccgcagcctaacaaa aagtcccttg tctttctgca 300 ataaatgctg acaccccctg gctttttaggactgacggct cacgatgcag ccgttgcgat 360 aattaattga caattacccg cacattgatgcatacttggc ggtcaggtca ggtcaggctg 420 aagcatacct attgggtcat ttatttgccgatcgtggtga aaagaatgca agtgataact 480 agttacgagt cgctttatga aagatggttggtcgaaactg tcaatatggc atgggcggca 540 aatcgtttgg tctcaactct atagcatgtactataattgg tcttttcatc acagtcacgc 600 caaagtgcca gtctcagact atggaccaaccactttcctc cttcacgtct aaattgactt 660 gatcaccaga ctcgaatatt ttttcttttcttctataccc ctaggatcat acaatacgaa 720 ccccaactca actcgagaga gagagtccccttcccaacat tttgacagcc cttgctcttc 780 tcctcccagg atgtaacaga agctgaaagggtacccctgt agcccacctt tacccaccat 840 cttttccatc tgtatcggtg catcccatcacaaccctcac gtggtccgag atcgtcgtta 900 cccgtattgg aagctcactc cgggcccaacgagagattgg accaaggaaa aataactttg 960 agacctcttc aagcagtcgg tcattcgttactgggatgtg tagtcgataa tgcggggtga 1020 caggccctca atccagcacc caccatcatgggcactgact gtactaccgg agcccatcat 1080 ttcgtttttg ggtcctggcg tctacttgaccgactgagtt tgccaagatg gatggcatga 1140 gagacagtgg ttaggctggg cgggtattgtgatgagagaa agcgagagac tagttagaag 1200 caaagaaaaa agatatataa gctgtcacatccctcatgaa catgctgttc ttgtaagtcg 1260 ggatatcagg gccagcttca gtattcagtatcctttctga gggagttgca ccttgtcaca 1320 gcttgtctgt ctatcactta tacttacccttggaccacgt tctttgtctg tcaagatgca 1380 tctcctatca ctcctctcaa ttgccacccttgcggtagcc agccctctga gcgttgaaga 1440 ttacgccaag gctctcgatg aaagaggtaaaacgattctc tgttcccata acaattccaa 1500 tactcacaga cctagctgtt tctgtctctaccaacgactt tggcaacttc aagttctaca 1560 tccagcacgg tgccgcagca tactgtaactctgaagccgc agccggtgca aaggtcacct 1620 gcggaggaaa cggttgccca acggtccagtccaatggtgc caccatcgtg gcatctttcc 1680 tgtaagtcta acatatcaca aacacatcatcaactccaaa cttacaaatc tctttatagt 1740 ggctcaaaga ctggcatcgg tggctacgtcgcgaccgact ctgcacgcaa ggaaatcgtc 1800 ctctcggttc gcggtagcac caacattcgcaactggctta ccaacctcga cttcgaccag 1860 gatgactgca gcttgacctc cggctgtggagtgcacggag gcttccagag agcctggaat 1920 gagatctcgg ccgcagcaac cgccgctgtcgcaaaggccc gcaaggcgaa cccttcgttc 1980 aaggtcgttg ccacaggtca ctcgttgggtggtgctgtag ctacactagc aggtgcgaac 2040 ctgcgagttg gtggtacgcc agttgacatctacacctacg gctcaccccg agttggaaac 2100 acgcaactcg ctgccttcat ctctaaccaggctggtggag agttccgcgt tacgaacgcc 2160 aaggaccccg tgcctcgtct cccccctctggtctttggat accggcacac atcccccgag 2220 tactggttgt ctggtagcgg aggtaacaaggttgactaca ccatcaatga tgtcaaggtg 2280 tgtgagggtg ctgccaacct tcagtgcaacggtggaacac tcggattgga tatcgacgcc 2340 catctccact acttccagga gaccgatgcttgctctggtt ccggtatcgc gtggagaaga 2400 tacaggagtg ctaagcgtga gagcatctcggagagggcca ctatgacaga tgccgagctg 2460 gagaagaagc ttaacaacta tgttgcgatggataaggagt atgtcaagac tcacgccaac 2520 cgctcatcgt agtatgacat ttacgcagtaacgatataat taccataaca aaaactctgg 2580 ataccattct ggtgcaagca tggcgaagaaaacatcatta tctatgtgaa tgtatcataa 2640 ccatccttac gccatgccgt tgatcttactactgagacaa aatactcagt catgtacaac 2700 aaactccaaa gcaccgaatg acttctggctttttggcaaa gcacgaaacc aatcattcaa 2760 acccctccac gaccatgccc tgcgcattgggaacacccac gagaatgaca ccacgaggca 2820 cgcggacact cttcaccttc atgcacccaaagacattgac ttcccggata ttagggcatg 2880 ctcggaaaat ggaacccaga acaaaatccgtcactgcctc acagaaactg atctccaatt 2940 2 349 PRT Fusarium venenatum 2 MetHis Leu Leu Ser Leu Leu Ser Ile Ala Thr Leu Ala Val Ala Ser 1 5 10 15Pro Leu Ser Val Glu Asp Tyr Ala Lys Ala Leu Asp Glu Arg Ala Val 20 25 30Ser Val Ser Thr Asn Asp Phe Gly Asn Phe Lys Phe Tyr Ile Gln His 35 40 45Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Ala Ala Gly Ala Lys Val 50 55 60Thr Cys Gly Gly Asn Gly Cys Pro Thr Val Gln Ser Asn Gly Ala Thr 65 70 7580 Ile Val Ala Ser Phe Leu Gly Ser Lys Thr Gly Ile Gly Gly Tyr Val 85 9095 Ala Thr Asp Ser Ala Arg Lys Glu Ile Val Leu Ser Val Arg Gly Ser 100105 110 Thr Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Asp Gln Asp Asp115 120 125 Cys Ser Leu Thr Ser Gly Cys Gly Val His Gly Gly Phe Gln ArgAla 130 135 140 Trp Asn Glu Ile Ser Ala Ala Ala Thr Ala Ala Val Ala LysAla Arg 145 150 155 160 Lys Ala Asn Pro Ser Phe Lys Val Val Ala Thr GlyHis Ser Leu Gly 165 170 175 Gly Ala Val Ala Thr Leu Ala Gly Ala Asn LeuArg Val Gly Gly Thr 180 185 190 Pro Val Asp Ile Tyr Thr Tyr Gly Ser ProArg Val Gly Asn Thr Gln 195 200 205 Leu Ala Ala Phe Ile Ser Asn Gln AlaGly Gly Glu Phe Arg Val Thr 210 215 220 Asn Ala Lys Asp Pro Val Pro ArgLeu Pro Pro Leu Val Phe Gly Tyr 225 230 235 240 Arg His Thr Ser Pro GluTyr Trp Leu Ser Gly Ser Gly Gly Asn Lys 245 250 255 Val Asp Tyr Thr IleAsn Asp Val Lys Val Cys Glu Gly Ala Ala Asn 260 265 270 Leu Gln Cys AsnGly Gly Thr Leu Gly Leu Asp Ile Asp Ala His Leu 275 280 285 His Tyr PheGln Glu Thr Asp Ala Cys Ser Gly Ser Gly Ile Ala Trp 290 295 300 Arg ArgTyr Arg Ser Ala Lys Arg Glu Ser Ile Ser Glu Arg Ala Thr 305 310 315 320Met Thr Asp Ala Glu Leu Glu Lys Lys Leu Asn Asn Tyr Val Ala Met 325 330335 Asp Lys Glu Tyr Val Lys Thr His Ala Asn Arg Ser Ser 340 345 3 35 DNAFusarium venenatum 3 cagtgaattg gcctcgatgg ccgcggccgc gaatt 35 4 35 DNAFusarium venenatum 4 aattcgcggc cgcggccatc gaggccaatt cactg 35 5 34 DNAFusarium venenatum 5 cacgaaggaa agacgatggc tttcacggtg tctg 34 6 34 DNAFusarium venenatum 6 cagacaccgt gaaagccatc gtctttcctt cgtg 34 7 46 DNAFusarium venenatum 7 ctatctcttc accatggtac cttaattaaa taccttgttg gaagcg46 8 46 DNA Fusarium venenatum 8 cgcttccaac aaggtattta attaaggtaccatggtgaag agatag 46 9 37 DNA Fusarium venenatum 9 cgttctttgt ctgtcagcatgcatctccta tcactcc 37 10 45 DNA Fusarium venenatum 10 ccagagtttttgttatggtt aattaatatc gttactgcgt aaatg 45

What is claimed is:
 1. An isolated polypeptide having lipase activity,selected from the group consisting of: (a) a polypeptide having an aminoacid sequence which has at least 85% identity with amino acids 31 to 350for the mature polypeptide of SEQ ID NO:2; (b) a polypeptide which isencoded by a nucleic acid sequence which hybridizes under highstringency conditions with (i) nucleotides 1525 to 2530 of SEQ ID NO:1,(ii) the cDNA sequence contained in nucleotides 1525 to 2530 of SEQ IDNO:1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or(iv) a complementary strand of (i), (ii), or (iii); (c) a variant of thepolypeptide having an amino acid sequence of SEQ ID NO:2 comprising asubstitution, deletion, and/or insertion of one or more amino acids; and(d) a fragment of (a) or (b), that has lipase activity.
 2. Thepolypeptide of claim 1, having an amino acid sequence which has at least85% identity with amino acids 31 to 350 of SEQ ID NO:2.
 3. Thepolypeptide of claim 2, having an amino acid sequence which has at least90% identity with amino acids 31 to 350 of SEQ ID NO:2.
 4. Thepolypeptide of claim 3, having an amino acid sequence which has at least95% identity with amino acids 31 to 350 of SEQ ID NO:2.
 5. Thepolypeptide of claim 4, having an amino acid sequence which has at least97% identity with amino acids 31 to 350 of SEQ ID NO:2.
 6. Thepolypeptide of any of claims 1-5, comprising the amino acid sequence ofSEQ ID NO:2.
 7. The polypeptide of any of claims 1-6, consisting of theamino acid sequence of SEQ ID NO:2 or a fragment thereof.
 8. Thepolypeptide of claim 7, consisting of the amino acid sequence of SEQ IDNO:2.
 9. The polypeptide of claim 8, which consists of amino acids 31 to350 of SEQ ID NO:2.
 10. The polypeptide of claim 1, which is encoded bya nucleic acid sequence which hybridizes under high stringencyconditions with (i) nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) thecDNA sequence contained in nucleotides 1525 to 2530 of SEQ ID NO:1,(iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv)a complementary strand of (i), (ii), or (iii).
 11. The polypeptide ofclaim 10, which is encoded by a nucleic acid sequence which hybridizesunder very high stringency conditions with (i) nucleotides 1525 to 2530of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 1525 to2530 of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii). 12.The polypeptide of claim 1, wherein the polypeptide is a variant of thepolypeptide having an amino acid sequence of SEQ ID NO:2 comprising asubstitution, deletion, and/or insertion of one or more amino acids. 13.The polypeptide of claim 1, which is encoded by the nucleic acidsequence contained in plasmid pEJG60 which is contained in E. coli NRRLB-30333.
 14. The polypeptide of any of claims 1-13 which has at least20% of the lipase activity of SEQ ID NO:2.
 15. An isolated nucleic acidsequence comprising a nucleic acid sequence which encodes thepolypeptide of any of claims 1-14.
 16. An isolated nucleic acid sequencecomprising a nucleic acid sequence having at least one mutation in themature polypeptide coding sequence of SEQ ID NO:1, in which the mutantnucleic acid sequence encodes a polypeptide consisting of amino acids 31to 350 of SEQ ID NO:2.
 17. An isolated nucleic acid sequence produced by(a) hybridizing a DNA under high stringency conditions with (i)nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) the cDNA sequencecontained in nucleotides 1525 to 2530 of SEQ ID NO:1, (iii) asubsequence of (i) or (ii) of at least 100 nucleotides, or (iv) acomplementary strand of (i), (ii), or (iii); and (b) isolating thenucleic acid sequence.
 18. The isolated nucleic acid sequence of claim17 produced by (a) hybridizing a DNA under very high stringencyconditions with (i) nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) thecDNA sequence contained in nucleotides 1525 to 2530 of SEQ ID NO:1,(iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv)a complementary strand of (i), (ii), or (iii); and (b) isolating thenucleic acid sequence.
 19. A nucleic acid construct comprising thenucleic acid sequence of claim 15 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.
 20. A recombinant expression vector comprising thenucleic acid construct of claim
 19. 21. A recombinant host cellcomprising the nucleic acid construct of claim
 19. 22. A method forproducing a mutant nucleic acid sequence, comprising (a) introducing atleast one mutation into the mature polypeptide coding sequence of SEQ IDNO:1, wherein the mutant nucleic acid sequence encodes a polypeptideconsisting of amino acids 31 to 350 of SEQ ID NO:2; and (b) recoveringthe mutant nucleic acid sequence.
 23. A mutant nucleic acid sequenceproduced by the method of claim
 22. 24. A method for producing apolypeptide, comprising (a) cultivating a strain comprising the mutantnucleic acid sequence of claim 23 encoding the polypeptide to produce asupernatant comprising the polypeptide; and (b) recovering thepolypeptide.
 25. A method for producing the polypeptide of any of claims1-14 comprising (a) cultivating a strain under conditions suitable forproduction of the polypeptide; and (b) recovering the polypeptide.
 26. Amethod for producing the polypeptide of any of claims 1-14 comprising(a) cultivating a host cell comprising a nucleic acid constructcomprising a nucleic acid sequence encoding the polypeptide underconditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 27. A method for producing a polypeptidecomprising (a) cultivating a host cell under conditions conducive forproduction of the polypeptide, wherein the host cell comprises a mutantnucleic acid sequence having at least one mutation in the maturepolypeptide coding sequence of SEQ ID NO:1, wherein the mutant nucleicacid sequence encodes a polypeptide consisting of amino acids 31 to 350of SEQ ID NO:2, and (b) recovering the polypeptide.
 28. A method forproducing the polypeptide of any of claims 1-14 comprising (a)cultivating a homologously recombinant cell, having incorporated thereina new transcription unit comprising a regulatory sequence, an exon,and/or a splice donor site operably linked to a second exon of anendogenous nucleic acid sequence encoding the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 29. A method for producing a mutant of acell, which comprises disrupting or deleting a nucleic acid sequenceencoding the polypeptide of any of claims 1-14 or a control sequencethereof, which results in the mutant producing less of the polypeptidethan the cell.
 30. A mutant produced by the method of claim
 29. 31. Themutant of claim 30, which further comprises a nucleic acid sequenceencoding a heterologous protein.
 32. A method for producing aheterologous polypeptide comprising (a) cultivating the mutant of claim31 under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 33. A nucleic acid construct comprising agene encoding a protein operably linked to one or both of a firstnucleic acid sequence encoding a signal peptide consisting ofnucleotides 1376 to 1420 of SEQ ID NO:1 and a second nucleic acidsequence encoding a propeptide consisting of nucleotides 1421 to 1465 ofSEQ ID NO:1, wherein the gene is foreign to the first and second nucleicacid sequences.
 34. A recombinant expression vector comprising thenucleic acid construct of claim
 33. 35. A recombinant host cellcomprising the nucleic acid construct of claim
 33. 36. A method forproducing a protein comprising (a) cultivating the recombinant host cellof claim 35 under conditions suitable for production of the protein; and(b) recovering the protein.
 37. A detergent composition comprising asurfactant and the polypeptide of any of claims 1-14.